FBXO22 promotes osteosarcoma progression via regulation of FOXO1 for ubiquitination and degradation

Abstract Accumulating evidence has demonstrated that F‐box protein 22 (FBXO22) participates in tumour development and progression in various types of human malignancies. However, the functions and detailed molecular mechanisms of FBXO22 in osteosarcoma tumorigenesis and progression remain elusive. In this study, we aimed to determine the effects of FBXO22 on the cell proliferation, migration and invasion of osteosarcoma cells using cell counting kit‐8 and Matrigel Transwell approaches. Moreover, we explored the molecular mechanisms by which FBXO22 mediated oncogenesis and progression in osteosarcoma via Western blotting, immunoprecipitation and ubiquitination. We found that FBXO22 depletion inhibited the proliferation, migration and invasion of osteosarcoma cells, whereas FBXO22 overexpression increased the proliferation and motility of osteosarcoma cells. Mechanistically, FBXO22 promoted the ubiquitination and degradation of FoxO1 in osteosarcoma cells. FBXO22 depletion reduced cell proliferation and motility via regulation of FoxO1. Taken together, our findings provide new insight into FBXO22‐induced osteosarcoma tumorigenesis. The inhibition of FBXO22 could be a promising strategy for the treatment of osteosarcoma.

carcinogenesis by targeting their specific substrates. 14,15Accumulating evidence has indicated that F-box protein 22 (FBXO22) participates in tumour development and progression in various types of human malignancies. 16,17High FBXO22 expression is correlated with poor overall survival in numerous cancers. 18One study revealed that FBXO22 facilitates hepatocellular carcinoma (HCC) progression by regulating Kruppel-like transcription factor 4 for destabilization. 19Another study reported that FBXO22 targets p21 for degradation and enhances HCC development. 20In addition, depletion of FBXO22 retarded cell invasion, migration and angiogenesis via modulation of the hypoxia inducible factor 1 subunit alpha and vascular endothelial growth factor (VEGF) pathways in melanoma. 21In lung cancer cells, FBXO22 overexpression inactivates liver kinase B1 and enhances lung cancer cell growth. 22Similarly, nuclear factor erythroid-related factor 2 accelerated tumour metastasis via the suppression of FBXO22-mediated degradation of BTB domain and CNC homologue 1 in lung cancer. 23 osteosarcoma cells, Hou and co-workers reported that the ln-cRNA small nucleolar RNA host gene 14 (SNHG14) accelerated tumour progression by targeting miR-433-3p and FBXO22 in osteosarcoma. 24ockdown of FBXO22 inhibited the proliferation and motility of 143B and SaOS-2 osteosarcoma cells. 24Nevertheless, the function and detailed molecular mechanisms of FBXO22 are elusive in osteosarcoma, and further studies are needed to determine whether FBXO22 might be a potent target for therapy in osteosarcoma patients.Therefore, in this study, we aimed to determine the effects of FBXO22 on the proliferation, migration and invasion of osteosarcoma cells.Moreover, we explored the molecular mechanisms by which FBXO22 mediated oncogenesis and progression in osteosarcoma.

| Cell culture
The U2OS and MG63 osteosarcoma cell lines were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum (FBS) and 100 U/mL penicillin, and 100 μg/mL streptomycin for cell culture.SaOS-2 cells were maintained in McCoy's 5A modified medium supplemented with 10% FBS, and 100 U/mL penicillin and 100 μg/mL streptomycin.The cells were cultured in 5% CO 2 at 37°C.All human cell lines had been authenticated using STR (or SNP) profiling within the last 3 years.All experiments were performed with mycoplasma-free cells.

| Quantitative real-time reverse transcription-PCR
The transfected cells were harvested, and TRIzol reagent was used to extract total RNA.The cDNA reverse transcription kit was subsequently used to reverse transcribe the mRNA to cDNA according to the manufacturer's manual.Polymerase chain reaction (PCR) was carried out using the SYBR Green PCR Master Mix Kit as described previously. 25

| Western blotting assay
The treated osteosarcoma cells were lysed with lysis buffer.The bicinchoninic acid (BCA) method was subsequently used to determine the protein concentration.Protein expression was analysed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and an immunostaining assay was performed according to previous publications. 26The following antibodies were used: mouse anti-FBXO22 (1:1000, sc-100,736, Santa Cruz), mouse anti-Ub (1:1000, sc-166,355, Santa Cruz) and anti-tubulin (1:5000, T9028, Sigma-Aldrich).ImageJ software was used to obtain the quantitative results. 27

| Transfection assays
The osteosarcoma cells were cultured in 6-well plates overnight.The cells were then transfected with various plasmids using Lipofectamine 2000.The FBXO22 short hairpin RNA (shRNA), FBXO22 cDNA plasmid and control vectors were purchased from GenePharma Company (Shanghai, China).Cell viability, invasion and migration were assessed after transfection for different durations as described previously. 28

| CCK-8 assay
Cell viability was measured using the cell counting kit-8 (CCK-8) approach as described previously. 29The transfected osteosarcoma cells were cultured in 96-well plates for different durations.The cells were subsequently maintained at 37°C for 3-4 h after addition of 10 μL of CCK-8 agent.A microplate reader was used to measure the OD values at 450 nm.

| EdU assay
The 5-ethynyl-2-deoxyuridine (EdU) assay was used to detect cell proliferation because EdU can be incorporated into replicating DNA to affect DNA synthesis.The transfected osteosarcoma cells were seeded into 96-well plates for different durations.The cells were incubated with EdU for 2 h and fixed with 4% formaldehyde for 30 min.
Next, the cells were stained with Hoechst 33342 for 30 min.EdUpositive cells were photographed by fluorescence microscopy.The percentage of viable cells was calculated based on the numbers of EdU-stained and Hoechst 33342-stained cells. 30

| Wound healing assay
A wound healing assay was used to determine directional cell migration.The transfected osteosarcoma cells were cultured in 6-well plates until the confluence reached more than 90%.A 100 μL pipette tip was then used to establish a wound in the cell monolayer at the centre of the plate.After the cells were washed and the cell debris was removed with PBS, the wound area was captured at different times.ImageJ software was used to measure the wound closure distance. 31

| Transwell migration and invasion assays
The transfected osteosarcoma cells were cultured on the 24-well inserts on the top layers.The membrane of the top layer contained Matrigel for the invasion assay or no Matrigel for the migration assay.
The top layers were supplemented with 200 μL of FBS-free medium, while the bottom layers were supplemented with 500 μL of medium supplemented with 10% FBS.At different incubation times, the migratory and invasive cells that migrated through the membrane were stained and photographed under a microscope. 32

| Protein half-life assays
The protein half-life of the transfected cells was analysed.Briefly, osteosarcoma cells were treated with 100 μg/mL cycloheximide for different durations.The cells were subsequently harvested, and protein expression levels were detected via Western blotting. 33

| Immunoprecipitation assay
The osteosarcoma cells were transfected with different plasmids for 20 hours.Then, 10 μM MG132 was added to the cells, which were incubated for 10 hours.The cells were lysed in Immunoprecipitation (IP) lysis buffer.BCA reagent was used to measure the protein concentration.Then, 10 mg of cell lysate was incubated with the primary antibody-conjugated beads for 4 hours in a cold room.After the immunocomplexes were washed, Western blotting was performed with the indicated antibodies. 34

| In vivo ubiquitination
The osteosarcoma cells were transfected with different plasmids, including His-Ub.Next, the cells were treated with 10 μM MG132 for 10 h, followed by washing and lysis in IP lysis buffer.Then, 10 mg of the cell lysate was incubated with the primary FoxO1 antibody for 4 h in a cold room.The cell lysate was subsequently incubated with protein A/G plus agarose overnight.Beads were detected by Western blotting to measure ubiquitination. 35

| Mouse xenograft assay
Six-week-old BALB/c0nu/nu mice were used for the xenograft assays.
Transfected osteosarcoma cells were collected and injected into the nude mice.A Vernier calliper was used to measure the longest and shortest diameters of the tumours every 4 days.The tumour volumes were calculated using the following formula: L × W 2 × 0.52, where L represents the longest diameter and W represents the shortest diameter.
After 32 days, the mice were sacrificed, and the tumours were photographed and weighed.The animal studies were approved by the Animal Experimentation Ethical Committee of China Medical University.

| Statistical analysis
The results are presented as the means ± standard deviations compared with the control group.Statistical analysis of multiple groups was performed via two-way analysis of variance with Tukey's post hoc test.Differences between two groups were compared using student's t-test.A p value <0.05 was considered to indicate statistical significance.GraphPad Prism software was used for all statistical analyses.

| FBXO22 depletion inhibits the proliferation of osteosarcoma cells
To explore the effect of FBXO22 on the proliferation of osteosarcoma cells, we downregulated the expression of FBXO22 in SaOS-2 and U2OS cells.The RT-PCR data revealed that FBXO22 mRNA levels were decreased in SaOS-2 and U2OS cells after FBXO22 shRNA transfection (Figure 1A).Moreover, we performed Western blotting analysis to determine the knockdown efficacy of FBXO22 shRNAs.We found that FBXO22 expression was downregulated in the shFBXO22treated groups (Figure 1B).A CCK-8 assay was conducted to detect the proliferation of osteosarcoma cells after FBXO22 downregulation.
The CCK-8 data revealed that silencing FBXO22 attenuated the proliferation of SaOS-2 and U2OS cells (Figure 1C).To confirm that cell proliferation was affected by FBXO22 knockdown, an EdU assay was performed in osteosarcoma cells after FBXO22 depletion.Knockdown of FBXO22 reduced the number of EdU-positive SaOS-2 and U2OS cells (Figure 1D).Taken together, these findings indicated that depletion of FBXO22 inhibited the proliferation of osteosarcoma cells.

| FBXO22 depletion reduces invasion and migration
To determine the effect of FBXO22 on the migratory and invasive ability of osteosarcoma cells, we performed wound healing assay and Transwell migration and invasion assays in osteosarcoma cells.Wound healing assays revealed that the closure rate in U2OS and SaOS-2 cells was attenuated in the shFBXO22-transfected groups compared with the scramble groups (Figure 2A).Transwell migration assays revealed that, compared with scramble cells,

| FBXO22 overexpression increases the proliferation and motility of osteosarcoma cells
Next, we upregulated the expression of FBXO22 in osteosarcoma cells via FBXO22 cDNA transfection.The Western blotting analysis revealed that FBXO22 expression was markedly increased in the FBXO22 cDNA-transfected U2OS, SaOS-2 and MG63 cells compared with their empty vector (EV)-transfected counterparts (Figure 3A).The CCK-8 assay data revealed that, compared with EV transfection, FBXO22 cDNA transfection increased the proliferation of U2OS and SaOS-2 cells (Figure 3B).EdU assay data suggested that the overexpression of FBXO22 increased the number of EdUpositive U2OS, SaOS-2 and MG63 cells, indicating that the upregulation of FBXO22 promoted the proliferation of osteosarcoma cells (Figure 3C,D).Transwell migration assays revealed that upregulation of FBXO22 enhanced the migratory ability of osteosarcoma cells (Figure 2D).In addition, Transwell invasion assay data confirmed that increased FBXO22 promoted the invasiveness of osteosarcoma cells (Figure 4A,B).Furthermore, the results of the wound healing assay revealed that overexpression of FBXO22 accelerated the rate of wound closure in osteosarcoma cells (Figure 4C).Therefore, FBXO22 overexpression promoted the proliferation, migration and invasion of osteosarcoma cells.

| FBXO22 promotes the ubiquitination and degradation of FoxO1
To determine whether FoxO1 is the substrate of FBXO22, we performed a Western blotting analysis in osteosarcoma cells after FBXO22 depletion or overexpression.We observed that depletion of FBXO22 increased the expression of FoxO1 at the protein level in osteosarcoma cells (Figure 5A).Consistently, FBXO22 overexpression reduced FoxO1 protein levels in osteosarcoma cells (Figure 5B).The RT-PCR data indicated that depletion of FBXO22 did not alter the expression of FoxO1 at the mRNA level (Figure 5C).To determine whether FBXO22 interacted with FoxO1, we conducted an IP assay in 293 T and U2OS cells.IP data indicated that FBXO22 could bind to FoxO1 in 293 T and U2OS cells (Figure 5D).Moreover, in vivo ubiquitination data clearly revealed that overexpression of FBXO22 enhanced the ubiquitination of FoxO1 in 293 T and U2OS cells (Figure 5E).Furthermore, MG132 treatment blocked FoxO1 accumulation in osteosarcoma cells after cotransfection with Myc-FBXO22 and Flag-FoxO1 (Figure 5F).In addition, half-life assays demonstrated that FBXO22 knockdown prolonged the half-life of FoxO1 in U2OS and SaOS-2 cells (Figure 6A,B).
These results suggested that FBXO22 governed the protein stability of FoxO1 in a posttranslational manner.

| FBXO22 depletion reduces cell proliferation via regulation of FoxO1
To verify whether FBXO22 knockdown inhibited cell proliferation via the regulation of FoxO1 stability, we cotransfected U2OS and SaOS-2 cells with shFBXO22 or shFoxO1.We observed that shF-BXO22 transfection increased the expression of FoxO1 protein, which partly abolished the shFoxO1-induced inhibition of FoxO1 in osteosarcoma cells (Figure 6C).The results of the CCK-8 assay revealed that downregulation of FoxO1 increased the proliferation of U2OS and SaOS-2 cells (Figure 6D).Knockdown of FBXO22 inhibited the proliferation of osteosarcoma cells, which was abrogated by shFoxO1 transfection (Figure 6D).Similar results were obtained via the EdU assay, which suggested that shFBXO22 reduced the proliferation of osteosarcoma cells and that this phenotype was reversed by shFoxO1 transfection (Figure 6E).Taken together, these findings indicated that the downregulation of FBXO22 repressed osteosarcoma cell proliferation by targeting the FoxO1 pathway.

| FBXO22 depletion reduces cell motility by targeting FoxO1
To test whether depletion of FBXO22 affected cell motility by regulating FoxO1 in osteosarcoma cells, we performed Transwell migration and invasion assays in U2OS and SaOS-2 cells after shFBXO22 transfection in combination with shFoxO1 treatment.Transwell Furthermore, depletion of FoxO1 accelerated the invasive ability of U2OS and SaOS-2 cells (Figure 7C).Consistent with these findings, FBXO22 knockdown reduced the invasive ability of osteosarcoma cells, which was abolished by shFoxO1 treatment (Figure 7C).These results suggested that FBXO22 depletion reduced cell motility by targeting FoxO1 in osteosarcoma cells.

| FBXO22 knockdown inhibits tumorigenesis in mice
Our in vitro results showed that depletion of FBXO22 reduced the proliferation of osteosarcoma cells.To validate this finding, we used nude mice to determine whether FBXO22 depletion in SaOS-2 cells retarded tumour growth in vivo.We observed that FBXO22 knockdown inhibited the growth of tumour xenografts in mice (Figure 7D).Tumour weights and volumes were decreased in the FBXO22 depletion group (Figure 7E,F).Moreover, we conducted Western blotting analysis to measure the expression of FoxO1 and FBXO22 in xenograft tumours.We observed that FoxO1 expression was upregulated in FBXO22-knockdown xenograft tumour tissues (Figure 7G).Therefore, FBXO22 knockdown inhibited tumour growth in mice.

| DISCUSS ION
In the present study, we reported that FBXO22 plays a tumourpromoting role in osteosarcoma cells via regulating the abundance of the FoxO1 protein.Recently, Lin et al. reported that FBXO22 targets p57 for ubiquitination and subsequent degradation and enhances cervical cancer progression. 25In cervical cancer cells, ectopic expression of FBXO22 promotes the viability of cervical cancer cells in vitro and facilitates tumour growth in vivo.Ectopic F I G U R E 5 F-box protein 22 (FBXO22) targets FoxO1 for ubiquitination and degradation.(A) Western blotting analysis was performed to measure the protein levels of FBXO22 and FoxO1 in SaOS-2 and U2OS cells after shFbxo22 transfection for 72 h.(B) Western blotting analysis was performed to measure the protein levels of FBXO22 and FoxO1 in osteosarcoma cells after Fbxo22 cDNA transfection for 72 h.(C) RT-PCR was performed to measure the FoxO1 mRNA levels in SaOS-2 and U2OS cells after shFbxo22 transfection for 72 h.(D) Immunoblotting (IB) of immunoprecipitates and whole-cell lysates (WCLs) was conducted to detect the interaction between FBXO22 and FoxO1.(E) IB analysis of WCLs and productions of ubiquitination derived from U2OS cells transfected with different constructs.(F) IB analysis of WCLs derived from U2OS and SaOS-2 cells transfected with different constructs.MG132 (10 μM) was used to treat the cells before harvesting.expression of FBXO22 attenuates cell apoptosis and facilitates G1/S phase progression in cervical cancer cells. 25In A549 non-small cell lung cancer (NSCLC) cells, overexpression of FBXO22 increases cell proliferation via the promotion of cyclin-dependent kinase 4 protein levels and the regulation of phosphatase and tensin homologue (PTEN). 18In epithelial ovarian cancer cells, FBXO22 facilitates metastasis and proliferation and represses autophagy by regulating the mitogen-activated protein kinase (MAPK) and extracellular signalregulated kinase (ERK) pathway. 36  Consistent with these reports, we demonstrated that FBXO22 could be an oncoprotein involved in osteosarcoma tumorigenesis and progression.
FoxO1, a transcription factor, has been identified as a tumour suppressor in a variety of human cancers. 38One study showed that liver X receptor α suppressed the proliferation of osteosarcoma cells via the promotion of FoxO1 expression. 39Another study revealed that miR-135b could accelerate cell proliferation and invasion via the inhibition of FoxO1 in osteosarcoma cells. 40Similarly, miR-374a downregulates the expression of FoxO1 and promotes cell proliferation promotion in human osteosarcoma. 41Additionally, miR-196a enhances cell growth and reduces apoptosis by targeting the PTEN/ Akt/FoxO1 pathway in osteosarcoma. 42FoxO1 blocks osteosarcoma tumorigenesis through suppression of the Wnt/β-catenin signalling pathway. 43In our study, we found that inhibition of FoxO1 enhanced the proliferation, migration and invasion of osteosarcoma cells.FBXO22 exhibited oncogenic functions in osteosarcoma cells via a reduction in the FoxO1 protein.
Interestingly, several studies have reported that FBXO22 plays an antitumor role in multiple cancer types. 44,45For example, FBXO22 reduces tumour metastasis via the suppression of matrix metalloproteinase-9 (MMP-9)-involved invasion and migration and the blockade of VEGF-induced angiogenesis in renal cell carcinoma. 45In fact, FBXO22 does not affect the proliferation of renal cancer cells; however, FBXO22 restricts the invasion and migration of renal cancer cells by abolishing epithelial-mesenchymal transition (EMT) and increasing the activity of tissue inhibitor of matrix metalloproteinase-1 (TIMP-1), leading to the suppression of MMP-9 expression and activity.Moreover, FBXO22 reduces the secretion of VEGF and impairs tube formation in cells.Li et al. reported that FBXO22 blocks tumour metastasis via modification of lysine demethylase 5A (KDM5A) ubiquitin and modulates H3K4me3 demethylation in triple-negative breast cancer (TNBC). 44FBXO22 attenuates KDM5A expression via ubiquitination, leading to a reduction in H3K4me3 demethylation and upregulation of p16, which inhibits oncogenesis and metastasis in TNBC. 44FBXO22 increases the cisplatin sensitivity of tumour cells by mediating the ubiquitination and degradation of CD147 in A549 lung cancer cells. 46FBXO22 targets the degradation of the oncoprotein NSD2 in acute lymphoblastic leukaemia cells. 47A CRISPR activation screen revealed that FBXO22 degrades endogenous proteins, such as bromodomain-containing protein 4 (BRD4) and enchinoderm microtubule-associated protein-like 4-anaplastic lymphoma kinase (EML4-ALK). 48Alkylamine-tethered molecules were found to be required for the recruitment of FBXO22 to degrade FKBP12 protein. 49These reports indicate that FBXO22 performs its biological functions in a cancer type-dependent manner.Surprisingly, FBXO22 exhibited both antimetastatic functions and protumorigenic effects on breast cancer development and progression. 50Another study revealed that FBXO22-induced degradation of KDM4B influences the activity of selective oestrogen receptor modulators (SERMs) in breast cancer. 51Moreover, FBXO22 inhibited tumour cell invasion and metastasis by controlling human homologue of mouse double minute 2 (HDM2) degradation in breast cancer. 52These reports suggest a complicated role of FBXO22 in breast cancer progression.Recently, FBXO22 was reported to be critically involved in immunotherapy by targeting programmed death-ligand 1 (PD-L1) in tumour cells. 53FBXO22 promotes PD-L1 degradation via an ubiquitination and leads to increased tumour cell sensitivity to DNA damage in NSCLC.Cyclin-dependent kinase 5 (CDK5) has been shown to increase the expression of PD-L1 in NSCLC and medulloblastoma. 53Silencing of CDK5 increases FBXO22 expression and subsequently reduces PD-L1 expression in NSCLC.This study revealed that blockade of both CDK5 and PD-L1 could increase the efficacy of immunotherapy in NSCLC. 53e group reported that FoxO1 could interact with and promote the expression of PD-1 in CD8 + T cells during chronic infection. 54nce immunotherapies are useful for osteosarcoma treatment, 55 targeting FBXO22 could be an ideal approach for treating osteosarcoma.However, the relationships among FBXO22, PD-1, PD-L1 and FoxO1 in osteosarcoma should be clarified for future immunotherapy application.
FBXO22 was also shown to be regulated by several regulatory factors, such as p53, 56 the lncRNA SNHG14 24 and circ_0006282. 57searchers have revealed that noncoding RNAs play essential roles in carcinogenesis, including in osteosarcoma. 58He and colleagues have reported that circ_0006282 sponges miR-155 and elevates FBXO22 expression, contributing to gastric cancer progression. 57There is a need to determine the mechanisms responsible for the regulation of FBXO22 in osteosarcoma.Several critical limitations should be mentioned.For example, numerous downstream targets of FBXO22, such as p57, PTEN and HDM2, have been identified.It is unclear whether FBXO22 targets the degradation of p57, PTEN and HDM2 in osteosarcoma, which requires future exploration.However, whether FBXO22 affects the proliferation and motility of osteosarcoma cells by targeting FoxO1 and other proteins, such as p57, PTEN and MMP-9, is elusive.The associations between FBXO22 and clinical features should be determined in patients with osteosarcoma.Determining the correlation between FBXO22 and FoxO1 in osteosarcoma tissues is critical.Moreover, the development of inhibitors of FBXO22 for the treatment of osteosarcoma is pivotal.In summary, this is the first study to identify FoxO1 as a novel ubiquitin substrate of FBXO22.
shFBXO22-transfected cells displayed a reduced migratory ability (Figure 2B,C).Moreover, the Transwell invasion assay results demonstrated a decreased invasive capacity of shFBXO22-transfected F I G U R E 1 Silencing of F-box protein 22 (FBXO22) inhibits the proliferation, migration and invasion of osteosarcoma cells.(A) RT-PCR was performed to measure the FBXO22 mRNA levels in SaOS-2 and U2OS cells after shFbxo22 transfection for 72 h.(B) Western blotting analysis was performed to measure the protein levels of FBXO22 in SaOS-2 and U2OS cells after shFbxo22 transfection for 72 h.(C) A CCK-8 assay was conducted to measure the viability of SaOS-2 and U2OS cells after shFbxo22 transfection for different durations.(D) An EdU assay was performed to measure the proliferation of SaOS-2 and U2OS cells after shFbxo22 transfection for 72 h.**p < 0.01, ***p < 0.001, ****p < 0.0001 versus control.F I G U R E 2 Silencing of F-box protein 22 (FBXO22) inhibits the migration and invasion of osteosarcoma cells.(A) A wound healing assay was utilized to measure the rate of wound closure in SaOS-2 and U2OS cells after shFbxo22 transfection for 20 h.(B) Transwell migration and invasion assays were used to detect the migratory and invasive ability of SaOS-2 and U2OS cells after shFbxo22 transfection for 20 h.(C) Quantitative data are shown in Panel B. **p < 0.01, ***p < 0.001 versus control.osteosarcoma cells compared with the scramble group (Figure 2B,C).Taken together, these findings indicated that depletion of FBXO22 suppressed the migratory and invasive ability of osteosarcoma cells.
protein 22 (FBXO22) overexpression promotes the proliferation of osteosarcoma cells.(A) Western blotting analysis was performed to measure the protein levels of FBXO22 in osteosarcoma cells after Fbxo22 cDNA transfection for 72 h.(B) A CCK-8 assay was conducted to measure the viability of SaOS-2 and U2OS cells after FBXO22 cDNA transfection for different durations.***p < 0.001 versus control.(C) An EdU assay was performed to measure the proliferation of osteosarcoma cells after FBXO22 cDNA transfection for 72 h.(D) Right panel: Quantitative data are shown for the EdU assay.***p < 0.001 versus control.

F I G U R E 4 F
-box protein 22 (FBXO22) overexpression promotes the migration and invasion of osteosarcoma cells.(A) Transwell migration and invasion assays were used to detect the migratory and invasive ability of osteosarcoma cells after FBXO22 cDNA transfection for 20 h.(B) Quantitative data are shown for Panel A. **p < 0.01, ***p < 0.001 versus control.(C) A wound healing assay was used to measure the rate of wound closure in osteosarcoma cells after FBXO22 cDNA transfection for 10 h.migration assays revealed that shFoxO1 treatment enhanced the migratory ability of U2OS and SaOS-2 cells (Figure 7A).Moreover, the shFBXO22-induced inhibition of cell migration was abrogated by shFoxO1 treatment in osteosarcoma cells (Figure 7A,B).

F I G U R E 6
Silencing of F-box protein 22 (FBXO22) inhibits proliferation by regulating FoxO1.(A) Western blotting analysis was performed to measure the half-life of FoxO1 protein in SaOS-2 cells after shFbxo22 transfection for different durations.(B) Quantitative data are shown for the half-life of FoxO1.(C) Western blotting analysis was performed to measure the protein levels of FoxO1 in SaOS-2 and U2OS cells after shFbxo22 transfection plus shFoxO1 transfection.(D) A CCK-8 assay was conducted to measure the viability of SaOS-2 and U2OS cells after shFbxo22 transfection plus shFoxO1 transfection for different durations.(E) Left panel: An EdU assay was performed to measure the proliferation of SaOS-2 and U2OS cells after shFbxo22 transfection plus shFoxO1 transfection for 72 h.Right panel: Quantitative data are shown for the EdU assay.**p < 0.01, ***p < 0.001 versus control, # p < 0.05 versus shFBXO22 or shFoxO1 alone.F I G U R E 7 Silencing of F-box protein 22 (FBXO22) inhibits cell migration, invasion and tumour growth.(A) Left panel: Transwell migration assays were utilized to detect the migratory ability of SaOS-2 and U2OS cells after shFbxo22 transfection plus shFoxO1 transfection for 20 h.Right panel: Quantitative data are shown for left panel.(B) Quantitative data are shown for the migration ability of osteosarcoma cells.(C) Left panel: Transwell invasion assays were utilized to detect the invasive ability of SaOS-2 and U2OS cells after shFbxo22 transfection plus shFoxO1 transfection for 20 h.Right panel: Quantitative data are shown for left panel.*p < 0.05, **p < 0.01, ***p < 0.001 versus control, # p < 0.05 versus shFBXO22 or shFoxO1.(D) ShFBXO22-transfected SaOS-2 cells were injected subcutaneously into BALB/c-nu/nu mice.After 32 days, the tumours were dissected, and images were obtained.(E) The weights of the dissected tumours are shown.(F) The tumour volumes were measured over the different time periods.(G) Western blotting analysis was performed to measure the expression of FBXO22 and FoxO1 in the dissected tumours.***p < 0.001 versus control.

FBXO22
specifically interacted with FoxO1, and promoted its ubiquitination and subsequent degradation.Overexpression of FBXO22 promoted cell proliferation and migration and invasion.Our findings provide evidence for a novel mechanism by which FBXO22 promotes the degradation of the FoxO1 tumour suppressor in osteosarcoma in part.AUTH O R CO NTR I B UTI O N S He Zhang: Data curation (equal); formal analysis (equal); investigation (equal); methodology (equal); resources (equal); software (equal); writing -original draft (equal).Yang Bai: Data curation (equal); formal analysis (equal); investigation (equal); methodology (equal); resources (equal); software (equal); writing -original draft (equal).Jiatong Li: Formal analysis (supporting); investigation 37 et al. discovered that FBXO22targets nuclear PTEN for degradation to accelerate oncogenesis.37